The group I self-splicing introns, exemplified by the Tetrahymena ribosomal RNA intron, are RNA enzymes (ribozymes) that catalyze phosphodiester exchange reactions. This proposal is focused on the analysis of substrate binding by this class of RNA enzymes. Previous experiments have led to the identification of part of the guanosine binding site of this RNA enzyme. Here, methods for further defining the guanosine binding site are presented, and novel approaches to defining the RNA substrate binding site are described. These experiments use a recently described system for the in vitro genetic analysis of interactions within the ribozyme. Several enhancements of these in vitro selection and amplification methods are proposed, along with plans to use these techniques extensively in the study of ribozyme-substrate interactions. Potential enzymesubstrate interactions that are identified will be studied by the kinetic analysis of mutant enzymes in combination with chemically synthesized substrate analogue.